Cutting Edge
The
Journal of
Immunology
Cutting Edge: Identification and Characterization of Human Intrahepatic CD49a+ NK Cells Nicole Marquardt,* Vivien Be´ziat,* Sanna Nystro¨m,* Julia Hengst,† Martin A. Ivarsson,* Eliisa Keka¨la¨inen,* Helene Johansson,‡ Jenny Mjo¨sberg,* Magnus Westgren,x Tim O. Lankisch,† Heiner Wedemeyer,† Ewa C. Ellis,‡ Hans-Gustaf Ljunggren,* Jakob Michae¨lsson,* and Niklas K. Bjo¨rkstro¨m* Although NK cells are considered innate, recent studies in mice revealed the existence of a unique lineage of hepatic CD49a+DX52 NK cells with adaptive-like features. Development of this NK cell lineage is, in contrast to conventional NK cells, dependent on Tbet but not Eomes. In this study, we describe the identification of a T-bet+Eomes2CD49a+ NK cell subset readily detectable in the human liver, but not in afferent or efferent hepatic venous or peripheral blood. Human intrahepatic CD49a+ NK cells express killer cell Ig-like receptor and NKG2C, indicative of having undergone clonal-like expansion, are CD56bright, and express low levels of CD16, CD57, and perforin. After stimulation, CD49a+ NK cells express high levels of inflammatory cytokines but degranulate poorly. CD49a+ NK cells retain their phenotype after expansion in long-term in vitro cultures. These results demonstrate the presence of a likely human counterpart of mouse intrahepatic NK cells with adaptive-like features. The Journal of Immunology, 2015, 194: 2467–2471.
N
atural killer cells are an essential part of the innate immune system providing rapid responses against invading pathogens and cells undergoing malignant transformation (1). NK cell responses include cytotoxicity as well as production of cytokines, for example, IFN-g and TNF, that can fine-tune innate and adaptive immune responses (2). Recently, several studies have shown that mouse NK cells exhibit adaptive-like features, including recall responses to allergens and viral infections (3, 4). In particular, a subset of intrahepatic NK cells with a CD49a+DX52 phenotype has been attributed to these responses (4, 5). In the human set-
*Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden; †Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany; ‡Division of Transplantation Surgery, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden; and xCenter for Fetal Medicine, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden ORCID: 0000-0002-0967-076X (N.K.B.).
ting, discrete subsets of expanded NK cell populations have been found in peripheral blood after infection with CMV (6, 7), hantavirus (8), and Chikungunya virus (9). Much less is known about adaptive-like features of NK cells in human peripheral tissues; specifically, it is not known whether the human liver harbors a counterpart of the mouse CD49a+DX52 NK cell subset. NK cells originate from the bone marrow, and several transcription factors, including E4BP4, T-bet, and Eomes, control NK cell development (2). Recently, use of knockout and reporter systems disclosed the presence of distinct NK cell lineages with separate transcription factor expression patterns in mice (10–12). Conventional mouse NK cells developing in the bone marrow are dependent on E4BP4 and the downstream targets Eomes and Id2 (10, 11). In contrast, some tissue-resident mouse NK cell populations, including CD49a+DX52 intrahepatic NK cells, develop independently of E4BP4 and Eomes (12), but are dependent on T-bet (10). However, the transcriptional requirements for human conventional and tissue-resident NK cells remain poorly understood. In this study, we report the existence of human tissueresident intrahepatic CD49a+ NK cells. This subset of NK cells was phenotypically distinct from conventional NK (cNK) cells and non-NK innate lymphoid cells (ILCs). In contrast to intrahepatic cNK cells, the CD49a+ NK cells were T-bet+Eomes2 and retained this profile upon long-term expansion in vitro. Moreover, CD49a+ NK cells produced high levels of proinflammatory cytokines such as IFN-g, TNF, and GM-CSF. These data support the notion of T-bet–dependent extramedullary NK cell development, as previously shown in the mouse (10), and identify a putative human counterpart to intrahepatic CD49a+DX52 mouse NK cells. the Novo Nordisk Foundation, A˚ke Olsson’s Foundation, Jeansson’s Foundation, Bengt Ihre’s Foundation, Groschinsky’s Foundation, Hedlunds’ Foundation, and Julin’s Foundation. Address correspondence and reprint requests to Dr. Niklas K. Bjo¨rkstro¨m, Center for Infectious Medicine, F59, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden. E-mail address: niklas. [email protected] The online version of this article contains supplemental material.
Received for publication November 11, 2014. Accepted for publication January 9, 2015.
Abbreviations used in this article: cNK cell, conventional NK cell; ILC, innate lymphoid cell; KIR, killer cell Ig-like receptor.
This work was supported by the Swedish Research Council, the German Research Foundation, the Swedish Cancer Society, the Swedish Society for Medical Research, the Cancer Research Foundations of Radiumhemmet, the Swedish Society of Medicine,
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402756
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CUTTING EDGE: HUMAN INTRAHEPATIC CD49a+ NK CELLS
Materials and Methods
CMVpp65 stimulation assay
Preparation of human liver material
Liver mononuclear cells were thawed and distributed at a final concentration of 5 3 106 cells/ml in a 96-well U-bottom plate. Cells were stimulated or not with CMVpp65 overlapping peptides (JPT Peptide Technologies, 1 mg/ml for each peptide) in the presence of brefeldin A. After 16 h of incubation, the cells were stained for extracellular receptors, permeabilized, and stained for intracellular IFN-g. Donors were considered CMV+ when .0.1% of T cells produced IFN-g in response to CMVpp65 overlapping peptides.
Human adult liver tissue was obtained either from patients undergoing surgical liver resection for removal of primary or metastatic tumors or from organ donors whose livers were not used for transplantation. Written consent was provided and the regional Ethical Review Board in Stockholm, Sweden approved the study. Of tumor-bearing livers, only nonaffected, healthy tissues were used. Immune cells were isolated using a previously described protocol (13). Portal venous blood was drawn from the portal vein after laparotomy, and hepatic venous blood was drawn during wedge hepatic venous pressure measurements. Density gradient centrifugation was used to isolate leukocytes.
Flow cytometry Abs and clones against the following proteins were used: CD3 (UCHT1, PECy5, Beckman Coulter), CD7 (M-T701, Alexa Fluor 700, BioLegend, or Horizon V450, BD Biosciences), CD14 (M5E2, Horizon V500, BD Biosciences), CD16 (3G8, Brilliant Violet 711 or 785, BioLegend, or VEP13, PE, Miltenyi Biotec), CD19 (HIB19, Horizon V500, BD Biosciences), CD29 (MAR4, PE-Cy5, BD Biosciences), CD34 (581, ECD, Beckman Coulter), CD45 (HI30, Alexa Fluor 700, BioLegend), CD45RA (HI100, Brilliant Violet 785, BioLegend), CD49a (TS2A, PE, Alexa Fluor 647, BioLegend, or SR84, PE, BD Biosciences), CD56 (HCD56, Brilliant Violet 711, BioLegend, or NCAM16, PE-Cy7, BD Biosciences), CD57 (TB01, purified, eBioscience), CD69 (TP1.55.3, ECD, Beckman Coulter), CD103 (Ber-ACT8, PE-Cy7, BioLegend), CD107a (H4A3, FITC, BD Biosciences), CD117 (104D2D1, PE-Cy5.5, Beckman Coulter), CD127 (R34.34, PE-Cy7, Beckman Coulter), CD161 (HP-3G10, Brilliant Violet 605, BioLegend), DNAM-1 (DX11, FITC, BD Biosciences), GM-CSF (BVD2-21C11, PE-CF594, BD Biosciences), granzyme A (CB9, Pacific Blue, BioLegend), Granzyme B (GB11, PE-CF94, BD Biosciences), killer cell Ig-like receptor (KIR)2DL1 (143211, allophycocyanin, R&D Systems), KIR2DL1/S1 (EB6, PE-Cy5.5, Beckman Coulter), KIR2DL2/3/S2 (GL183, PE-Cy5.5, Beckman Coulter), KIR2DL3 (180701, FITC, R&D Systems), KIR3DL1 (DX9, Alexa Fluor 700, BioLegend), NKG2A (Z199, custom conjugate, allophycocyanin–Alexa Fluor 750, Beckman Coulter), NKG2C (134591, PE, R&D Systems), NKG2D (1D11, PE-CF594, BD Biosciences), NKp30 (p30-15, Alexa Fluor 647, BD Biosciences), NKp44 (P44-8, biotin, BioLegend), NKp46 (9E2, Brilliant Violet 421, BioLegend), perforin (dG9, FITC, eBioscience), Eomes (WF1928, FITC or eF660, eBioscience), T-bet (O4-46, CF-594, BD Biosciences), retinoic acid–related orphan receptor gt (AFKJS-9, PE, eBioscience), Helios (22F6, Pacific Blue, BioLegend), GATA-3 (TWAI, eF660, eBiosciences), IFN-g (4S.B3, Brilliant Violet 570, BioLegend), MIP-1b (D21-1351, PE, BD Biosciences), and TNF (Mab11, Pacific Blue, eBioscience). Purified NKG2C (134591, R&D Systems) was biotinylated using a Fluoreporter Mini-biotin-XX protein labeling kit (Life Technologies) and detected using streptavidin–Qdot 605 (Invitrogen). All samples were stained with Live/Dead Aqua or Green (Invitrogen) to discriminate live and dead cells. Samples were analyzed using a BD LSRFortessa flow cytometer equipped with four lasers. Data were analyzed using FlowJo 9.7.4 (Tree Star).
KIR and HLA genotyping KIR genotyping and KIR ligand determination were performed using PCRSSP technology with a KIR typing kit and a KIR HLA ligand kit (both Olerup SSP).
Functional NK cell assay Mononuclear liver cells were thawed and enriched for NK cells by magnetic cell sorting using an NK cell isolation kit (Miltenyi Biotec). Cells were resuspended in complete medium and rested overnight in the presence of 5 ng/ml IL-15 (PeproTech). The cells were then stimulated with PMA (10 ng/ml) and ionomycin (500 nM) for 4 h in the presence of monensin and brefeldin A for the last 3 h of incubation. Anti-human CD107a was present throughout the assay. Finally, cells were stained and analyzed by flow cytometry, as described above.
NK cell sorting and expansion assay Mononuclear liver cells were thawed and for some experiments CFSE labeled. Then, the cells were surface stained, followed by sorting on a FACSAria III into the following live Lin2CD32CD56+ NK cell subsets: CD49a+, CD49a2CD162, and CD49a2CD16+CD572. To avoid cross-linking of CD16, the nonagonistic antiCD16 clone VEP13 was used. After sorting, the NK cells were cocultured with irradiated 721.221 or transfected 721.221-AEH cells (HLA-E+) and HLA-C1+/ C2+ feeder PBMCs in expansion medium (5% human serum and 100 ng/ml IL15) for 3 wk. Afterward, cells were stained for flow cytometry analysis, and total cell counts were calculated using counting beads.
Results and+ Discussion 2 +
CD3 CD49a CD56 NK cells are present in human adult livers
Mouse livers contain a subset of CD49a+DX52 NK cells that under certain conditions exhibit adaptive-like immune features, such as potent recall responses to viruses and allergens (4, 5). This NK cell population is virtually absent in other organs. We hypothesized that a phenotypically similar subset of cells could be present in the human liver. To test this, we investigated enzymatically prepared liver perfusates from healthy human donors and from non–tumor-affected liver sections of patients with primary and metastatic liver malignancies. Strikingly, a subset of CD32 CD49a+CD56+ lymphocytes was identified in 12 of 29 livers examined. In the positive livers, this subset made up 0.11–12.7% (average 2.3%) of the total CD32CD56+ lymphocyte population (Fig. 1A). In contrast, very low frequencies of CD32CD49a+CD56+ cells were detectable in peripheral blood (Fig. 1A). Moreover, hardly any CD32CD49a+CD56+ lymphocytes were found in human first trimester fetal livers (Supplemental Fig. 1A). Taken together, we here identify a human phenotypically similar counterpart to mouse liver–resident CD49a+DX52 NK cells. Human intrahepatic CD32CD49a+CD56+ cells are bona fide NK cells with a T-bet+Eomes2 transcription factor profile
To further characterize human liver–resident CD32CD49a+CD56+ cells (hereafter referred to as CD49a+ NK cells), we first analyzed their phenotype in detail. These cells did not express CD127 (IL7Ra) (Supplemental Fig. 1B) or the transcription factors retinoic acid–related orphan receptor gt and Helios (data not shown), indicating that CD49a+ NK cells are distinct from non–NK ILCs (14, 15). The CD49a+ NK cells also lacked expression of NKp44 and expressed only low levels of CD103 (data not shown). This suggests that the cells are different in phenotype from the population of non–NK CD32CD56+ ILC1 recently reported to reside in human tonsils (15). Instead, the CD49a+ NK cells displayed a CD32CD7+NKp46+CD162CD56brightCD122low phenotype, which was, except for the low expression of CD122, similar to the profile of conventional CD32CD49a2CD56brightCD162 NK cells that represent ∼50% of total intrahepatic NK cells (Fig. 1B). The transcription factors E4BP4, T-bet, and Eomes are critical for cNK cell development and differentiation (2). However, mouse intrahepatic CD49a+DX52 NK cells develop independently of Eomes and E4BP4 but are dependent on T-bet (10, 12). Interestingly, the CD49a+ NK cells identified in human liver expressed high levels of T-bet but only low or undetectable levels of Eomes (Fig. 1C, Supplemental Fig. 1C). Hence, this profile was markedly different from that of intrahepatic Eomes+T-bet+/2 cNK cells, but similar to the mouse CD49a+DX52 NK cells. Thus, based on the cell surface phenotype and transcription factor expression, our data suggest that the CD49a+ NK cell population is a distinct subset of human bona fide intrahepatic NK cells sharing features with mouse CD49a+DX52 NK cells.
The Journal of Immunology
FIGURE 1. The human adult liver contains CD49a+ NK cells. (A) Staining of CD56 and CD49a on freshly isolated live CD45+CD32CD142CD192 lymphocytes from three adult liver samples (of 29 investigated) and one adult PBMC sample (of 15 investigated). (B) Stainings for NKp46, CD16, CD122, and CD7 on CD49a+ and CD49a2 intrahepatic CD56+CD32 cells. (C) Stainings for Eomes and T-bet expression on CD49a+, CD49a2CD162, and CD49a2CD16+ intrahepatic CD56+CD32 cells.
Human intrahepatic CD49a+ NK cells express KIRs but lack CD57 and NKG2A
Next, we sought to determine the differentiation status of the intrahepatic CD49a+ NK cells. On the basis of CD11b and CD27 staining, CD49a+DX52 mouse liver NK cells were reported to display a more immature phenotype as compared with conventional intrahepatic NK cells (5, 10). Human cNK cell differentiation is most readily assessed by the cell surface expression of CD56, NKG2A, CD57, and KIRs. In brief, more immature conventional peripheral blood NK cells are CD56brightNKG2A+CD572KIR2, whereas differentiation is associated with transition to CD56dim, loss of NKG2A, acquisition of CD57, and increasing expression of KIRs (16). Similar to peripheral blood-derived NK cells, both immature and mature subsets resided within intrahepatic cNK cells (Fig. 2A, Supplemental Fig. 1D). Interestingly, almost all CD49a+ intrahepatic NK cells were CD56bright and lacked expression of CD16, CD57, and NKG2A, but .80% of them expressed KIRs (Fig. 2A, Supplemental Fig. 1D), and as such did not follow the pattern of differentiation normally seen among cNK cells.
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noted. Finally, low levels of activating KIRs, such as KIR2DS1 and KIR2DS4, were coexpressed by some CD49a+ NK cells (data not shown). Taken together, the skewed KIR profile of the intrahepatic CD49a+ NK cells suggests this subset of cells to have undergone a clonal-like expansion. In addition to the particular expression pattern of KIRs, a vast majority of the human intrahepatic CD49a+ NK cells expressed the activating receptor NKG2C (Fig. 2A, Supplemental Figs. 1D, 2D). Abundant NKG2C expression on human peripheral blood NK cells has been linked to CMV seropositivity, as well as the expression of CD57 in conjunction with a single inhibitory self-HLA class I–binding KIR (6). Additionally, expansion of NKG2C+CD57+KIR+ NK cells occurs also during acute viral infections (8, 9). We did not have access to the CMV serostatus of liver donors in the present study. However, CMVpp65-specific intrahepatic CD8 T cells could be detected in three of three donors with a CD49a+ NK cell subset as well as in three of three donors without a CD49a+ subset (Supplemental Fig. 1E, 1F). This suggests that there is no strict correlation between CMV seropositivity and expansion of intrahepatic CD49a+ NK cells. Furthermore, in several respects, the phenotype of the intrahepatic CD49a+ NK cell subset differed markedly from that of the expanded NKG2C+ NK cell population observed in peripheral blood following CMV infection or reactivation. This included the lack of CD16 and CD57 as described above, low expression of CD45RA, Eomes, and perforin (Fig. 3A, Supplemental Figs. 1G, 2D), as well as high expression of NKp30, NKG2D, and CD69 on the intrahepatic CD49a+ NK cells (Supplemental Figs. 1H, 2D). In contrast, peripheral blood NKG2C+ NK cell expansions are CD16+CD57+perforinhigh but express low levels of NKp30, NKG2D, and CD69 (Supplemental Fig. 2D) (6, 17).
Human intrahepatic CD49a+ NK cells display features of clonal-like expansion with specific patterns of KIRs
To further characterize KIR expression on the intrahepatic CD49a+ NK cells, we genotyped donors for KIR and HLA and performed a high-resolution KIR phenotyping of the cells by flow cytometry. Intriguingly, the CD49a+ NK cells displayed an oligoclonal KIR expression pattern with at least 50% (and in some cases up to 80%) of the cells having a distinct KIR profile (Fig. 2B). In some donors, a single inhibitory KIR dominated the profile, whereas in others a specific combination of two or three inhibitory KIRs was coexpressed by the CD49a+ cells (Fig. 2B). The profile of the CD49a+ NK cells was clearly distinct from that of the CD49a2CD16+ cNK cells of the same donor (Fig. 2B). Additionally, the oligoclonal KIR expression pattern of the CD49a+ NK cells was different between donors, and no clear dominance of a particular inhibitory KIR was
FIGURE 2. Human intrahepatic CD49a+ NK cells have an immature phenotype but express high levels of self-HLA class I–binding KIRs and NKG2C. (A) Stainings for NKG2A, CD57, panKIR2D, and NKG2C on freshly isolated CD49a+, CD49a2CD162, and CD49a2CD16 intrahepatic NK cells. (B) KIR and HLA genotyping (left) and expression patterns of the major inhibitory KIRs (right) on CD49a+ and CD492CD16+ intrahepatic NK cells from two representative liver samples of eight investigated.
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blood of the liver. Interestingly, neither source, that is, the afferent (portal venous) or efferent (hepatic venous) blood of the liver, contained a detectable CD49a+ NK cell population (Fig. 4A), suggesting that CD49a+ NK cells are largely liverresident. CD49a (integrin a1) partners with CD29 (integrin b1) on the surface of cells to form an a1b1 integrin complex and binds to collagen (18). Only CD49a+ cells expressed CD29 (Supplemental Fig. 2A), indicating that these cells may form a functional receptor. Because virus-specific CD8+ T cells are kept in nonlymphoid tissues during primary immune responses in a CD49a-dependent manner (18), it is plausible that CD49a has a similar role in retaining the CD49a+ NK cells in the liver parenchyma contributing to liver residency. FIGURE 3. Human intrahepatic CD49a+ NK cells produce high levels of cytokines. (A) Stainings for perforin, granzyme A, and granzyme B on CD49a+, CD49a2CD162, and CD49a2CD16+ intrahepatic NK cells. (B) Stainings and summary of data (C) for CD107a, IFN-g, TNF, GM-CSF, and MIP-1b in intrahepatic CD49a+ and CD49a2 NK cells with or without PMA/ionomycin stimulation for 4 h (n = 5). *p , 0.05, ***p , 0.001, Student t test.
Human intrahepatic CD49a+ NK cells produce high levels of proinflammatory cytokines upon stimulation
Having identified a new subset of intrahepatic NK cells, we next analyzed their functional properties. The CD49a+ NK cells expressed low levels of perforin and granzyme A as compared with conventional CD49a2 intrahepatic NK cells but, instead, contained high levels of granzyme B (Fig. 3A, Supplemental Fig. 1G). Furthermore, CD49a+ NK cells expressed high levels of the activating receptors NKp46, NKp30, NKG2D, and DNAM-1 (Fig. 1B, Supplemental Fig. 1H). The lack of CD16 expression and low expression of perforin and granzyme A indicated reduced cytotoxic capacity. Accordingly, upon stimulation with PMA/ionomycin, degranulation was weaker in the CD49a+ subset than in their CD49a2 NK cell counterparts (Fig. 3B, 3C). Despite this poor capacity for degranulation, polyclonal stimulation led to a significantly higher production of cytokines such as IFN-g, TNF, and GM-CSF in the CD49a+ subset as compared with CD49a2 NK cells (Fig. 3B, 3C). The capacity to produce high levels of IFN-g, TNF, and GM-CSF, as well as the poor degranulation response and low levels of cytotoxic effector molecules, is consistent with the functional profile of mouse intrahepatic CD49a+DX52 NK cells (5, 10). A key function of NKG2C+ NK cell expansions in peripheral blood is potent Ab-dependent cellular cytotoxicity capacity (6, 7). However, the human intrahepatic CD49a+ NK cells described in the present study lack expression of CD16 and, thus, Ab-dependent cellular cytotoxicity capacity. Taken together, intrahepatic CD49a+ NK cells produce high levels of cytokines, but they degranulated poorly as compared with cNK cells. Human CD49a+ NK cells are largely liver-resident
Parabiosis experiments have revealed that mouse intrahepatic CD49a+DX52 NK cells are liver-resident (5, 12). To investigate whether the human intrahepatic CD49a+ NK cells similarly showed signs of liver residency, we analyzed whether these cells could be detected in afferent or efferent venous
Human intrahepatic CD49a+ NK cells harbor a significant proliferative capacity and show a stable phenotype upon in vitro culture
Next, we assessed the proliferative capacity of intrahepatic CD49a+ NK cells. Highly purified CD49a+, CD49a2CD162, and CD49a2CD16+CD572 intrahepatic subsets of NK cells were stimulated for 3 wk with irradiated PBMCs as feeder cells, IL-15, and untransfected or HLA-E–transfected 721.221 cells. The CD49a+ subset expanded ∼800-fold during the 3 wk of stimulation (Fig. 4B). Additional stimulation with 721.221 cells or specifically through the NKG2C receptor via HLA-E did not further increase the proliferative response (Fig. 4B). Despite expressing low levels of CD122 (Fig. 1B), the CD49a+ cells
FIGURE 4. Human intrahepatic CD49a+ NK cells are tissue-resident and expand with retained phenotype upon stimulation. (A) Stainings for CD49a on NK cells from liver tissue, sinusoidal, portal venous, hepatic venous, and peripheral blood (n = 4–6). (B) Fold expansion of sorted CD49a+ intrahepatic NK cells after 3 wk of culture with feeder cells supplemented with IL-15, IL15 plus 721.221–wild-type cells, or IL-15 plus 721.221-AEH cells (HLA-E expressing) (n = 4, mean 6 SEM). (C) Frequency of cells positive for NKG2C and CD16 within sorted intrahepatic NK cell subsets ex vivo or after 3 wk of expansion with feeder cells and IL-15 (n = 4, mean 6 SEM). (D) Expression of perforin, T-bet, and Eomes after 3 wk of expansion with feeder cells and IL-15 (n = 4, mean 6 SEM). n.d., not detected.
The Journal of Immunology
expanded as efficiently as did the CD49a2 NK cell subsets investigated (data not shown). Furthermore, to determine whether intrahepatic CD49a+ NK cells represented an immature subset with the capacity to acquire more mature characteristics, or, alternatively, possessed a stable phenotype, we performed a phenotypic analysis of the cells following 3 wk of expansion. Intriguingly, the CD49a+ subset had a stable phenotype, including retained low expression of Eomes, perforin, NKG2A, and CD57, and high levels of T-bet and KIR (Fig. 4C, 4D, Supplemental Fig. 2E). In contrast, conventional CD49a2CD162 NK cells upregulated CD16, perforin, and T-bet upon proliferation (Fig. 4C, 4D). After 3 wk of culture, it was noted that sorted CD49a2 NK cells did upregulate CD49a. However, the level of expression was clearly lower as compared with the CD49+ NK cells characterized in this study (Supplemental Fig. 2B, 2C). Stimulating the CD49a+ NK cells additionally through NKG2C for 3 wk did not yield a difference in their phenotype (data not shown). On the contrary, peripheral blood NKG2C+ NK cells have been shown to respond vigorously upon NKG2C triggering (6). These data further corroborate the phenotypic (CD572CD162perforinlowEomes2) and functional (high cytokine production, low cytotoxicity) data presented above, indicating that expanded intrahepatic CD49a+ NK cells are a stable subset distinct from previously described peripheral blood NKG2C+ NK cell expansions. In conclusion, in this study we provide evidence for the existence of a previously unrecognized subset of CD49a+ NK cells in the human liver. These cells share phenotypic and functional traits with mouse intrahepatic tissue-resident CD49a+DX52 NK cells possessing adaptive-like features. To some extent, these human intrahepatic CD49a+ NK cells resemble the peripheral blood NKG2C+single KIR+ NK cell expansions reported in response to viruses. However, CD49a+ NK cells differ significantly in their transcription factor as well as effector molecule expression profiles, and in markers of differentiation. Taken together, these data support the notion of a T-bet–controlled extramedullary NK cell development in humans.
Disclosures The authors have no financial conflicts of interest.
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References 1. Vivier, E., E. Tomasello, M. Baratin, T. Walzer, and S. Ugolini. 2008. Functions of natural killer cells. Nat. Immunol. 9: 503–510. 2. Hesslein, D. G. T., and L. L. Lanier. 2011. Transcriptional control of natural killer cell development and function. Adv. Immunol. 109: 45–85. 3. Sun, J. C., J. N. Beilke, and L. L. Lanier. 2009. Adaptive immune features of natural killer cells. Nature 457: 557–561. 4. Paust, S., H. S. Gill, B.-Z. Wang, M. P. Flynn, E. A. Moseman, B. Senman, M. Szczepanik, A. Telenti, P. W. Askenase, R. W. Compans, and U. H. von Andrian. 2010. Critical role for the chemokine receptor CXCR6 in NK cellmediated antigen-specific memory of haptens and viruses. Nat. Immunol. 11: 1127–1135. 5. Peng, H., X. Jiang, Y. Chen, D. K. Sojka, H. Wei, X. Gao, R. Sun, W. M. Yokoyama, and Z. Tian. 2013. Liver-resident NK cells confer adaptive immunity in skin-contact inflammation. J. Clin. Invest. 123: 1444–1456. 6. Be´ziat, V., L. L. Liu, J.-A. Malmberg, M. A. Ivarsson, E. Sohlberg, A. T. Bjo¨rklund, C. Retie`re, E. Sverremark-Ekstro¨m, J. Traherne, P. Ljungman, et al. 2013. NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs. Blood 121: 2678–2688. 7. Foley, B., S. Cooley, M. R. Verneris, M. Pitt, J. Curtsinger, X. Luo, S. LopezVerge`s, L. L. Lanier, D. Weisdorf, and J. S. Miller. 2012. Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function. Blood 119: 2665–2674. 8. Bjo¨rkstro¨m, N. K., T. Lindgren, M. Stoltz, C. Fauriat, M. Braun, M. Evander, J. Michae¨lsson, K.-J. Malmberg, J. Klingstro¨m, C. Ahlm, and H.-G. Ljunggren. 2011. Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus. J. Exp. Med. 208: 13–21. 9. Petitdemange, C., P. Becquart, N. Wauquier, V. Be´ziat, P. Debre´, E. M. Leroy, and V. Vieillard. 2011. Unconventional repertoire profile is imprinted during acute chikungunya infection for natural killer cells polarization toward cytotoxicity. PLoS Pathog. 7: e1002268. 10. Daussy, C., F. Faure, K. Mayol, S. Viel, G. Gasteiger, E. Charrier, J. Bienvenu, T. Henry, E. Debien, U. A. Hasan, et al. 2014. T-bet and Eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow. J. Exp. Med. 211: 563–577. 11. Male, V., I. Nisoli, T. Kostrzewski, D. S. J. Allan, J. R. Carlyle, G. M. Lord, A. Wack, and H. J. M. Brady. 2014. The transcription factor E4bp4/Nfil3 controls commitment to the NK lineage and directly regulates Eomes and Id2 expression. J. Exp. Med. 211: 635–642. 12. Sojka, D. K., B. Plougastel-Douglas, L. Yang, M. A. Pak-Wittel, M. N. Artyomov, Y. Ivanova, C. Zhong, J. M. Chase, P. B. Rothman, J. Yu, et al. 2014. Tissueresident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells. eLife 3: e01659. 13. Lecluyse, E. L., and E. Alexandre. 2010. Isolation and culture of primary hepatocytes from resected human liver tissue. Methods Mol. Biol. 640: 57–82. 14. Spits, H., D. Artis, M. Colonna, A. Diefenbach, J. P. Di Santo, G. Eberl, S. Koyasu, R. M. Locksley, A. N. J. McKenzie, R. E. Mebius, et al. 2013. Innate lymphoid cells—a proposal for uniform nomenclature. Nat. Rev. Immunol. 13: 145–149. 15. Fuchs, A., W. Vermi, J. S. Lee, S. Lonardi, S. Gilfillan, R. D. Newberry, M. Cella, and M. Colonna. 2013. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-g-producing cells. Immunity 38: 769– 781. 16. Bjo¨rkstro¨m, N. K., P. Riese, F. Heuts, S. Andersson, C. Fauriat, M. A. Ivarsson, A. T. Bjo¨rklund, M. Flodstro¨m-Tullberg, J. Michae¨lsson, M. E. Rottenberg, et al. 2010. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood 116: 3853–3864. 17. Be´ziat, V., O. Dalgard, T. Asselah, P. Halfon, P. Bedossa, A. Boudifa, B. Hervier, I. Theodorou, M. Martinot, P. Debre´, et al. 2012. CMV drives clonal expansion of NKG2C+ NK cells expressing self-specific KIRs in chronic hepatitis patients. Eur. J. Immunol. 42: 447–457. 18. Ray, S. J., S. N. Franki, R. H. Pierce, S. Dimitrova, V. Koteliansky, A. G. Sprague, P. C. Doherty, A. R. de Fougerolles, and D. J. Topham. 2004. The collagen binding a1b1 integrin VLA-1 regulates CD8 T cell-mediated immune protection against heterologous influenza infection. Immunity 20: 167–179.
The
Journal of
Immunology
Cutting Edge: Identification and Characterization of Human Intrahepatic CD49a+ NK Cells Nicole Marquardt,* Vivien Be´ziat,* Sanna Nystro¨m,* Julia Hengst,† Martin A. Ivarsson,* Eliisa Keka¨la¨inen,* Helene Johansson,‡ Jenny Mjo¨sberg,* Magnus Westgren,x Tim O. Lankisch,† Heiner Wedemeyer,† Ewa C. Ellis,‡ Hans-Gustaf Ljunggren,* Jakob Michae¨lsson,* and Niklas K. Bjo¨rkstro¨m* Although NK cells are considered innate, recent studies in mice revealed the existence of a unique lineage of hepatic CD49a+DX52 NK cells with adaptive-like features. Development of this NK cell lineage is, in contrast to conventional NK cells, dependent on Tbet but not Eomes. In this study, we describe the identification of a T-bet+Eomes2CD49a+ NK cell subset readily detectable in the human liver, but not in afferent or efferent hepatic venous or peripheral blood. Human intrahepatic CD49a+ NK cells express killer cell Ig-like receptor and NKG2C, indicative of having undergone clonal-like expansion, are CD56bright, and express low levels of CD16, CD57, and perforin. After stimulation, CD49a+ NK cells express high levels of inflammatory cytokines but degranulate poorly. CD49a+ NK cells retain their phenotype after expansion in long-term in vitro cultures. These results demonstrate the presence of a likely human counterpart of mouse intrahepatic NK cells with adaptive-like features. The Journal of Immunology, 2015, 194: 2467–2471.
N
atural killer cells are an essential part of the innate immune system providing rapid responses against invading pathogens and cells undergoing malignant transformation (1). NK cell responses include cytotoxicity as well as production of cytokines, for example, IFN-g and TNF, that can fine-tune innate and adaptive immune responses (2). Recently, several studies have shown that mouse NK cells exhibit adaptive-like features, including recall responses to allergens and viral infections (3, 4). In particular, a subset of intrahepatic NK cells with a CD49a+DX52 phenotype has been attributed to these responses (4, 5). In the human set-
*Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden; †Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany; ‡Division of Transplantation Surgery, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden; and xCenter for Fetal Medicine, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden ORCID: 0000-0002-0967-076X (N.K.B.).
ting, discrete subsets of expanded NK cell populations have been found in peripheral blood after infection with CMV (6, 7), hantavirus (8), and Chikungunya virus (9). Much less is known about adaptive-like features of NK cells in human peripheral tissues; specifically, it is not known whether the human liver harbors a counterpart of the mouse CD49a+DX52 NK cell subset. NK cells originate from the bone marrow, and several transcription factors, including E4BP4, T-bet, and Eomes, control NK cell development (2). Recently, use of knockout and reporter systems disclosed the presence of distinct NK cell lineages with separate transcription factor expression patterns in mice (10–12). Conventional mouse NK cells developing in the bone marrow are dependent on E4BP4 and the downstream targets Eomes and Id2 (10, 11). In contrast, some tissue-resident mouse NK cell populations, including CD49a+DX52 intrahepatic NK cells, develop independently of E4BP4 and Eomes (12), but are dependent on T-bet (10). However, the transcriptional requirements for human conventional and tissue-resident NK cells remain poorly understood. In this study, we report the existence of human tissueresident intrahepatic CD49a+ NK cells. This subset of NK cells was phenotypically distinct from conventional NK (cNK) cells and non-NK innate lymphoid cells (ILCs). In contrast to intrahepatic cNK cells, the CD49a+ NK cells were T-bet+Eomes2 and retained this profile upon long-term expansion in vitro. Moreover, CD49a+ NK cells produced high levels of proinflammatory cytokines such as IFN-g, TNF, and GM-CSF. These data support the notion of T-bet–dependent extramedullary NK cell development, as previously shown in the mouse (10), and identify a putative human counterpart to intrahepatic CD49a+DX52 mouse NK cells. the Novo Nordisk Foundation, A˚ke Olsson’s Foundation, Jeansson’s Foundation, Bengt Ihre’s Foundation, Groschinsky’s Foundation, Hedlunds’ Foundation, and Julin’s Foundation. Address correspondence and reprint requests to Dr. Niklas K. Bjo¨rkstro¨m, Center for Infectious Medicine, F59, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden. E-mail address: niklas. [email protected] The online version of this article contains supplemental material.
Received for publication November 11, 2014. Accepted for publication January 9, 2015.
Abbreviations used in this article: cNK cell, conventional NK cell; ILC, innate lymphoid cell; KIR, killer cell Ig-like receptor.
This work was supported by the Swedish Research Council, the German Research Foundation, the Swedish Cancer Society, the Swedish Society for Medical Research, the Cancer Research Foundations of Radiumhemmet, the Swedish Society of Medicine,
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402756
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Materials and Methods
CMVpp65 stimulation assay
Preparation of human liver material
Liver mononuclear cells were thawed and distributed at a final concentration of 5 3 106 cells/ml in a 96-well U-bottom plate. Cells were stimulated or not with CMVpp65 overlapping peptides (JPT Peptide Technologies, 1 mg/ml for each peptide) in the presence of brefeldin A. After 16 h of incubation, the cells were stained for extracellular receptors, permeabilized, and stained for intracellular IFN-g. Donors were considered CMV+ when .0.1% of T cells produced IFN-g in response to CMVpp65 overlapping peptides.
Human adult liver tissue was obtained either from patients undergoing surgical liver resection for removal of primary or metastatic tumors or from organ donors whose livers were not used for transplantation. Written consent was provided and the regional Ethical Review Board in Stockholm, Sweden approved the study. Of tumor-bearing livers, only nonaffected, healthy tissues were used. Immune cells were isolated using a previously described protocol (13). Portal venous blood was drawn from the portal vein after laparotomy, and hepatic venous blood was drawn during wedge hepatic venous pressure measurements. Density gradient centrifugation was used to isolate leukocytes.
Flow cytometry Abs and clones against the following proteins were used: CD3 (UCHT1, PECy5, Beckman Coulter), CD7 (M-T701, Alexa Fluor 700, BioLegend, or Horizon V450, BD Biosciences), CD14 (M5E2, Horizon V500, BD Biosciences), CD16 (3G8, Brilliant Violet 711 or 785, BioLegend, or VEP13, PE, Miltenyi Biotec), CD19 (HIB19, Horizon V500, BD Biosciences), CD29 (MAR4, PE-Cy5, BD Biosciences), CD34 (581, ECD, Beckman Coulter), CD45 (HI30, Alexa Fluor 700, BioLegend), CD45RA (HI100, Brilliant Violet 785, BioLegend), CD49a (TS2A, PE, Alexa Fluor 647, BioLegend, or SR84, PE, BD Biosciences), CD56 (HCD56, Brilliant Violet 711, BioLegend, or NCAM16, PE-Cy7, BD Biosciences), CD57 (TB01, purified, eBioscience), CD69 (TP1.55.3, ECD, Beckman Coulter), CD103 (Ber-ACT8, PE-Cy7, BioLegend), CD107a (H4A3, FITC, BD Biosciences), CD117 (104D2D1, PE-Cy5.5, Beckman Coulter), CD127 (R34.34, PE-Cy7, Beckman Coulter), CD161 (HP-3G10, Brilliant Violet 605, BioLegend), DNAM-1 (DX11, FITC, BD Biosciences), GM-CSF (BVD2-21C11, PE-CF594, BD Biosciences), granzyme A (CB9, Pacific Blue, BioLegend), Granzyme B (GB11, PE-CF94, BD Biosciences), killer cell Ig-like receptor (KIR)2DL1 (143211, allophycocyanin, R&D Systems), KIR2DL1/S1 (EB6, PE-Cy5.5, Beckman Coulter), KIR2DL2/3/S2 (GL183, PE-Cy5.5, Beckman Coulter), KIR2DL3 (180701, FITC, R&D Systems), KIR3DL1 (DX9, Alexa Fluor 700, BioLegend), NKG2A (Z199, custom conjugate, allophycocyanin–Alexa Fluor 750, Beckman Coulter), NKG2C (134591, PE, R&D Systems), NKG2D (1D11, PE-CF594, BD Biosciences), NKp30 (p30-15, Alexa Fluor 647, BD Biosciences), NKp44 (P44-8, biotin, BioLegend), NKp46 (9E2, Brilliant Violet 421, BioLegend), perforin (dG9, FITC, eBioscience), Eomes (WF1928, FITC or eF660, eBioscience), T-bet (O4-46, CF-594, BD Biosciences), retinoic acid–related orphan receptor gt (AFKJS-9, PE, eBioscience), Helios (22F6, Pacific Blue, BioLegend), GATA-3 (TWAI, eF660, eBiosciences), IFN-g (4S.B3, Brilliant Violet 570, BioLegend), MIP-1b (D21-1351, PE, BD Biosciences), and TNF (Mab11, Pacific Blue, eBioscience). Purified NKG2C (134591, R&D Systems) was biotinylated using a Fluoreporter Mini-biotin-XX protein labeling kit (Life Technologies) and detected using streptavidin–Qdot 605 (Invitrogen). All samples were stained with Live/Dead Aqua or Green (Invitrogen) to discriminate live and dead cells. Samples were analyzed using a BD LSRFortessa flow cytometer equipped with four lasers. Data were analyzed using FlowJo 9.7.4 (Tree Star).
KIR and HLA genotyping KIR genotyping and KIR ligand determination were performed using PCRSSP technology with a KIR typing kit and a KIR HLA ligand kit (both Olerup SSP).
Functional NK cell assay Mononuclear liver cells were thawed and enriched for NK cells by magnetic cell sorting using an NK cell isolation kit (Miltenyi Biotec). Cells were resuspended in complete medium and rested overnight in the presence of 5 ng/ml IL-15 (PeproTech). The cells were then stimulated with PMA (10 ng/ml) and ionomycin (500 nM) for 4 h in the presence of monensin and brefeldin A for the last 3 h of incubation. Anti-human CD107a was present throughout the assay. Finally, cells were stained and analyzed by flow cytometry, as described above.
NK cell sorting and expansion assay Mononuclear liver cells were thawed and for some experiments CFSE labeled. Then, the cells were surface stained, followed by sorting on a FACSAria III into the following live Lin2CD32CD56+ NK cell subsets: CD49a+, CD49a2CD162, and CD49a2CD16+CD572. To avoid cross-linking of CD16, the nonagonistic antiCD16 clone VEP13 was used. After sorting, the NK cells were cocultured with irradiated 721.221 or transfected 721.221-AEH cells (HLA-E+) and HLA-C1+/ C2+ feeder PBMCs in expansion medium (5% human serum and 100 ng/ml IL15) for 3 wk. Afterward, cells were stained for flow cytometry analysis, and total cell counts were calculated using counting beads.
Results and+ Discussion 2 +
CD3 CD49a CD56 NK cells are present in human adult livers
Mouse livers contain a subset of CD49a+DX52 NK cells that under certain conditions exhibit adaptive-like immune features, such as potent recall responses to viruses and allergens (4, 5). This NK cell population is virtually absent in other organs. We hypothesized that a phenotypically similar subset of cells could be present in the human liver. To test this, we investigated enzymatically prepared liver perfusates from healthy human donors and from non–tumor-affected liver sections of patients with primary and metastatic liver malignancies. Strikingly, a subset of CD32 CD49a+CD56+ lymphocytes was identified in 12 of 29 livers examined. In the positive livers, this subset made up 0.11–12.7% (average 2.3%) of the total CD32CD56+ lymphocyte population (Fig. 1A). In contrast, very low frequencies of CD32CD49a+CD56+ cells were detectable in peripheral blood (Fig. 1A). Moreover, hardly any CD32CD49a+CD56+ lymphocytes were found in human first trimester fetal livers (Supplemental Fig. 1A). Taken together, we here identify a human phenotypically similar counterpart to mouse liver–resident CD49a+DX52 NK cells. Human intrahepatic CD32CD49a+CD56+ cells are bona fide NK cells with a T-bet+Eomes2 transcription factor profile
To further characterize human liver–resident CD32CD49a+CD56+ cells (hereafter referred to as CD49a+ NK cells), we first analyzed their phenotype in detail. These cells did not express CD127 (IL7Ra) (Supplemental Fig. 1B) or the transcription factors retinoic acid–related orphan receptor gt and Helios (data not shown), indicating that CD49a+ NK cells are distinct from non–NK ILCs (14, 15). The CD49a+ NK cells also lacked expression of NKp44 and expressed only low levels of CD103 (data not shown). This suggests that the cells are different in phenotype from the population of non–NK CD32CD56+ ILC1 recently reported to reside in human tonsils (15). Instead, the CD49a+ NK cells displayed a CD32CD7+NKp46+CD162CD56brightCD122low phenotype, which was, except for the low expression of CD122, similar to the profile of conventional CD32CD49a2CD56brightCD162 NK cells that represent ∼50% of total intrahepatic NK cells (Fig. 1B). The transcription factors E4BP4, T-bet, and Eomes are critical for cNK cell development and differentiation (2). However, mouse intrahepatic CD49a+DX52 NK cells develop independently of Eomes and E4BP4 but are dependent on T-bet (10, 12). Interestingly, the CD49a+ NK cells identified in human liver expressed high levels of T-bet but only low or undetectable levels of Eomes (Fig. 1C, Supplemental Fig. 1C). Hence, this profile was markedly different from that of intrahepatic Eomes+T-bet+/2 cNK cells, but similar to the mouse CD49a+DX52 NK cells. Thus, based on the cell surface phenotype and transcription factor expression, our data suggest that the CD49a+ NK cell population is a distinct subset of human bona fide intrahepatic NK cells sharing features with mouse CD49a+DX52 NK cells.
The Journal of Immunology
FIGURE 1. The human adult liver contains CD49a+ NK cells. (A) Staining of CD56 and CD49a on freshly isolated live CD45+CD32CD142CD192 lymphocytes from three adult liver samples (of 29 investigated) and one adult PBMC sample (of 15 investigated). (B) Stainings for NKp46, CD16, CD122, and CD7 on CD49a+ and CD49a2 intrahepatic CD56+CD32 cells. (C) Stainings for Eomes and T-bet expression on CD49a+, CD49a2CD162, and CD49a2CD16+ intrahepatic CD56+CD32 cells.
Human intrahepatic CD49a+ NK cells express KIRs but lack CD57 and NKG2A
Next, we sought to determine the differentiation status of the intrahepatic CD49a+ NK cells. On the basis of CD11b and CD27 staining, CD49a+DX52 mouse liver NK cells were reported to display a more immature phenotype as compared with conventional intrahepatic NK cells (5, 10). Human cNK cell differentiation is most readily assessed by the cell surface expression of CD56, NKG2A, CD57, and KIRs. In brief, more immature conventional peripheral blood NK cells are CD56brightNKG2A+CD572KIR2, whereas differentiation is associated with transition to CD56dim, loss of NKG2A, acquisition of CD57, and increasing expression of KIRs (16). Similar to peripheral blood-derived NK cells, both immature and mature subsets resided within intrahepatic cNK cells (Fig. 2A, Supplemental Fig. 1D). Interestingly, almost all CD49a+ intrahepatic NK cells were CD56bright and lacked expression of CD16, CD57, and NKG2A, but .80% of them expressed KIRs (Fig. 2A, Supplemental Fig. 1D), and as such did not follow the pattern of differentiation normally seen among cNK cells.
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noted. Finally, low levels of activating KIRs, such as KIR2DS1 and KIR2DS4, were coexpressed by some CD49a+ NK cells (data not shown). Taken together, the skewed KIR profile of the intrahepatic CD49a+ NK cells suggests this subset of cells to have undergone a clonal-like expansion. In addition to the particular expression pattern of KIRs, a vast majority of the human intrahepatic CD49a+ NK cells expressed the activating receptor NKG2C (Fig. 2A, Supplemental Figs. 1D, 2D). Abundant NKG2C expression on human peripheral blood NK cells has been linked to CMV seropositivity, as well as the expression of CD57 in conjunction with a single inhibitory self-HLA class I–binding KIR (6). Additionally, expansion of NKG2C+CD57+KIR+ NK cells occurs also during acute viral infections (8, 9). We did not have access to the CMV serostatus of liver donors in the present study. However, CMVpp65-specific intrahepatic CD8 T cells could be detected in three of three donors with a CD49a+ NK cell subset as well as in three of three donors without a CD49a+ subset (Supplemental Fig. 1E, 1F). This suggests that there is no strict correlation between CMV seropositivity and expansion of intrahepatic CD49a+ NK cells. Furthermore, in several respects, the phenotype of the intrahepatic CD49a+ NK cell subset differed markedly from that of the expanded NKG2C+ NK cell population observed in peripheral blood following CMV infection or reactivation. This included the lack of CD16 and CD57 as described above, low expression of CD45RA, Eomes, and perforin (Fig. 3A, Supplemental Figs. 1G, 2D), as well as high expression of NKp30, NKG2D, and CD69 on the intrahepatic CD49a+ NK cells (Supplemental Figs. 1H, 2D). In contrast, peripheral blood NKG2C+ NK cell expansions are CD16+CD57+perforinhigh but express low levels of NKp30, NKG2D, and CD69 (Supplemental Fig. 2D) (6, 17).
Human intrahepatic CD49a+ NK cells display features of clonal-like expansion with specific patterns of KIRs
To further characterize KIR expression on the intrahepatic CD49a+ NK cells, we genotyped donors for KIR and HLA and performed a high-resolution KIR phenotyping of the cells by flow cytometry. Intriguingly, the CD49a+ NK cells displayed an oligoclonal KIR expression pattern with at least 50% (and in some cases up to 80%) of the cells having a distinct KIR profile (Fig. 2B). In some donors, a single inhibitory KIR dominated the profile, whereas in others a specific combination of two or three inhibitory KIRs was coexpressed by the CD49a+ cells (Fig. 2B). The profile of the CD49a+ NK cells was clearly distinct from that of the CD49a2CD16+ cNK cells of the same donor (Fig. 2B). Additionally, the oligoclonal KIR expression pattern of the CD49a+ NK cells was different between donors, and no clear dominance of a particular inhibitory KIR was
FIGURE 2. Human intrahepatic CD49a+ NK cells have an immature phenotype but express high levels of self-HLA class I–binding KIRs and NKG2C. (A) Stainings for NKG2A, CD57, panKIR2D, and NKG2C on freshly isolated CD49a+, CD49a2CD162, and CD49a2CD16 intrahepatic NK cells. (B) KIR and HLA genotyping (left) and expression patterns of the major inhibitory KIRs (right) on CD49a+ and CD492CD16+ intrahepatic NK cells from two representative liver samples of eight investigated.
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blood of the liver. Interestingly, neither source, that is, the afferent (portal venous) or efferent (hepatic venous) blood of the liver, contained a detectable CD49a+ NK cell population (Fig. 4A), suggesting that CD49a+ NK cells are largely liverresident. CD49a (integrin a1) partners with CD29 (integrin b1) on the surface of cells to form an a1b1 integrin complex and binds to collagen (18). Only CD49a+ cells expressed CD29 (Supplemental Fig. 2A), indicating that these cells may form a functional receptor. Because virus-specific CD8+ T cells are kept in nonlymphoid tissues during primary immune responses in a CD49a-dependent manner (18), it is plausible that CD49a has a similar role in retaining the CD49a+ NK cells in the liver parenchyma contributing to liver residency. FIGURE 3. Human intrahepatic CD49a+ NK cells produce high levels of cytokines. (A) Stainings for perforin, granzyme A, and granzyme B on CD49a+, CD49a2CD162, and CD49a2CD16+ intrahepatic NK cells. (B) Stainings and summary of data (C) for CD107a, IFN-g, TNF, GM-CSF, and MIP-1b in intrahepatic CD49a+ and CD49a2 NK cells with or without PMA/ionomycin stimulation for 4 h (n = 5). *p , 0.05, ***p , 0.001, Student t test.
Human intrahepatic CD49a+ NK cells produce high levels of proinflammatory cytokines upon stimulation
Having identified a new subset of intrahepatic NK cells, we next analyzed their functional properties. The CD49a+ NK cells expressed low levels of perforin and granzyme A as compared with conventional CD49a2 intrahepatic NK cells but, instead, contained high levels of granzyme B (Fig. 3A, Supplemental Fig. 1G). Furthermore, CD49a+ NK cells expressed high levels of the activating receptors NKp46, NKp30, NKG2D, and DNAM-1 (Fig. 1B, Supplemental Fig. 1H). The lack of CD16 expression and low expression of perforin and granzyme A indicated reduced cytotoxic capacity. Accordingly, upon stimulation with PMA/ionomycin, degranulation was weaker in the CD49a+ subset than in their CD49a2 NK cell counterparts (Fig. 3B, 3C). Despite this poor capacity for degranulation, polyclonal stimulation led to a significantly higher production of cytokines such as IFN-g, TNF, and GM-CSF in the CD49a+ subset as compared with CD49a2 NK cells (Fig. 3B, 3C). The capacity to produce high levels of IFN-g, TNF, and GM-CSF, as well as the poor degranulation response and low levels of cytotoxic effector molecules, is consistent with the functional profile of mouse intrahepatic CD49a+DX52 NK cells (5, 10). A key function of NKG2C+ NK cell expansions in peripheral blood is potent Ab-dependent cellular cytotoxicity capacity (6, 7). However, the human intrahepatic CD49a+ NK cells described in the present study lack expression of CD16 and, thus, Ab-dependent cellular cytotoxicity capacity. Taken together, intrahepatic CD49a+ NK cells produce high levels of cytokines, but they degranulated poorly as compared with cNK cells. Human CD49a+ NK cells are largely liver-resident
Parabiosis experiments have revealed that mouse intrahepatic CD49a+DX52 NK cells are liver-resident (5, 12). To investigate whether the human intrahepatic CD49a+ NK cells similarly showed signs of liver residency, we analyzed whether these cells could be detected in afferent or efferent venous
Human intrahepatic CD49a+ NK cells harbor a significant proliferative capacity and show a stable phenotype upon in vitro culture
Next, we assessed the proliferative capacity of intrahepatic CD49a+ NK cells. Highly purified CD49a+, CD49a2CD162, and CD49a2CD16+CD572 intrahepatic subsets of NK cells were stimulated for 3 wk with irradiated PBMCs as feeder cells, IL-15, and untransfected or HLA-E–transfected 721.221 cells. The CD49a+ subset expanded ∼800-fold during the 3 wk of stimulation (Fig. 4B). Additional stimulation with 721.221 cells or specifically through the NKG2C receptor via HLA-E did not further increase the proliferative response (Fig. 4B). Despite expressing low levels of CD122 (Fig. 1B), the CD49a+ cells
FIGURE 4. Human intrahepatic CD49a+ NK cells are tissue-resident and expand with retained phenotype upon stimulation. (A) Stainings for CD49a on NK cells from liver tissue, sinusoidal, portal venous, hepatic venous, and peripheral blood (n = 4–6). (B) Fold expansion of sorted CD49a+ intrahepatic NK cells after 3 wk of culture with feeder cells supplemented with IL-15, IL15 plus 721.221–wild-type cells, or IL-15 plus 721.221-AEH cells (HLA-E expressing) (n = 4, mean 6 SEM). (C) Frequency of cells positive for NKG2C and CD16 within sorted intrahepatic NK cell subsets ex vivo or after 3 wk of expansion with feeder cells and IL-15 (n = 4, mean 6 SEM). (D) Expression of perforin, T-bet, and Eomes after 3 wk of expansion with feeder cells and IL-15 (n = 4, mean 6 SEM). n.d., not detected.
The Journal of Immunology
expanded as efficiently as did the CD49a2 NK cell subsets investigated (data not shown). Furthermore, to determine whether intrahepatic CD49a+ NK cells represented an immature subset with the capacity to acquire more mature characteristics, or, alternatively, possessed a stable phenotype, we performed a phenotypic analysis of the cells following 3 wk of expansion. Intriguingly, the CD49a+ subset had a stable phenotype, including retained low expression of Eomes, perforin, NKG2A, and CD57, and high levels of T-bet and KIR (Fig. 4C, 4D, Supplemental Fig. 2E). In contrast, conventional CD49a2CD162 NK cells upregulated CD16, perforin, and T-bet upon proliferation (Fig. 4C, 4D). After 3 wk of culture, it was noted that sorted CD49a2 NK cells did upregulate CD49a. However, the level of expression was clearly lower as compared with the CD49+ NK cells characterized in this study (Supplemental Fig. 2B, 2C). Stimulating the CD49a+ NK cells additionally through NKG2C for 3 wk did not yield a difference in their phenotype (data not shown). On the contrary, peripheral blood NKG2C+ NK cells have been shown to respond vigorously upon NKG2C triggering (6). These data further corroborate the phenotypic (CD572CD162perforinlowEomes2) and functional (high cytokine production, low cytotoxicity) data presented above, indicating that expanded intrahepatic CD49a+ NK cells are a stable subset distinct from previously described peripheral blood NKG2C+ NK cell expansions. In conclusion, in this study we provide evidence for the existence of a previously unrecognized subset of CD49a+ NK cells in the human liver. These cells share phenotypic and functional traits with mouse intrahepatic tissue-resident CD49a+DX52 NK cells possessing adaptive-like features. To some extent, these human intrahepatic CD49a+ NK cells resemble the peripheral blood NKG2C+single KIR+ NK cell expansions reported in response to viruses. However, CD49a+ NK cells differ significantly in their transcription factor as well as effector molecule expression profiles, and in markers of differentiation. Taken together, these data support the notion of a T-bet–controlled extramedullary NK cell development in humans.
Disclosures The authors have no financial conflicts of interest.
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References 1. Vivier, E., E. Tomasello, M. Baratin, T. Walzer, and S. Ugolini. 2008. Functions of natural killer cells. Nat. Immunol. 9: 503–510. 2. Hesslein, D. G. T., and L. L. Lanier. 2011. Transcriptional control of natural killer cell development and function. Adv. Immunol. 109: 45–85. 3. Sun, J. C., J. N. Beilke, and L. L. Lanier. 2009. Adaptive immune features of natural killer cells. Nature 457: 557–561. 4. Paust, S., H. S. Gill, B.-Z. Wang, M. P. Flynn, E. A. Moseman, B. Senman, M. Szczepanik, A. Telenti, P. W. Askenase, R. W. Compans, and U. H. von Andrian. 2010. Critical role for the chemokine receptor CXCR6 in NK cellmediated antigen-specific memory of haptens and viruses. Nat. Immunol. 11: 1127–1135. 5. Peng, H., X. Jiang, Y. Chen, D. K. Sojka, H. Wei, X. Gao, R. Sun, W. M. Yokoyama, and Z. Tian. 2013. Liver-resident NK cells confer adaptive immunity in skin-contact inflammation. J. Clin. Invest. 123: 1444–1456. 6. Be´ziat, V., L. L. Liu, J.-A. Malmberg, M. A. Ivarsson, E. Sohlberg, A. T. Bjo¨rklund, C. Retie`re, E. Sverremark-Ekstro¨m, J. Traherne, P. Ljungman, et al. 2013. NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs. Blood 121: 2678–2688. 7. Foley, B., S. Cooley, M. R. Verneris, M. Pitt, J. Curtsinger, X. Luo, S. LopezVerge`s, L. L. Lanier, D. Weisdorf, and J. S. Miller. 2012. Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function. Blood 119: 2665–2674. 8. Bjo¨rkstro¨m, N. K., T. Lindgren, M. Stoltz, C. Fauriat, M. Braun, M. Evander, J. Michae¨lsson, K.-J. Malmberg, J. Klingstro¨m, C. Ahlm, and H.-G. Ljunggren. 2011. Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus. J. Exp. Med. 208: 13–21. 9. Petitdemange, C., P. Becquart, N. Wauquier, V. Be´ziat, P. Debre´, E. M. Leroy, and V. Vieillard. 2011. Unconventional repertoire profile is imprinted during acute chikungunya infection for natural killer cells polarization toward cytotoxicity. PLoS Pathog. 7: e1002268. 10. Daussy, C., F. Faure, K. Mayol, S. Viel, G. Gasteiger, E. Charrier, J. Bienvenu, T. Henry, E. Debien, U. A. Hasan, et al. 2014. T-bet and Eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow. J. Exp. Med. 211: 563–577. 11. Male, V., I. Nisoli, T. Kostrzewski, D. S. J. Allan, J. R. Carlyle, G. M. Lord, A. Wack, and H. J. M. Brady. 2014. The transcription factor E4bp4/Nfil3 controls commitment to the NK lineage and directly regulates Eomes and Id2 expression. J. Exp. Med. 211: 635–642. 12. Sojka, D. K., B. Plougastel-Douglas, L. Yang, M. A. Pak-Wittel, M. N. Artyomov, Y. Ivanova, C. Zhong, J. M. Chase, P. B. Rothman, J. Yu, et al. 2014. Tissueresident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells. eLife 3: e01659. 13. Lecluyse, E. L., and E. Alexandre. 2010. Isolation and culture of primary hepatocytes from resected human liver tissue. Methods Mol. Biol. 640: 57–82. 14. Spits, H., D. Artis, M. Colonna, A. Diefenbach, J. P. Di Santo, G. Eberl, S. Koyasu, R. M. Locksley, A. N. J. McKenzie, R. E. Mebius, et al. 2013. Innate lymphoid cells—a proposal for uniform nomenclature. Nat. Rev. Immunol. 13: 145–149. 15. Fuchs, A., W. Vermi, J. S. Lee, S. Lonardi, S. Gilfillan, R. D. Newberry, M. Cella, and M. Colonna. 2013. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-g-producing cells. Immunity 38: 769– 781. 16. Bjo¨rkstro¨m, N. K., P. Riese, F. Heuts, S. Andersson, C. Fauriat, M. A. Ivarsson, A. T. Bjo¨rklund, M. Flodstro¨m-Tullberg, J. Michae¨lsson, M. E. Rottenberg, et al. 2010. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood 116: 3853–3864. 17. Be´ziat, V., O. Dalgard, T. Asselah, P. Halfon, P. Bedossa, A. Boudifa, B. Hervier, I. Theodorou, M. Martinot, P. Debre´, et al. 2012. CMV drives clonal expansion of NKG2C+ NK cells expressing self-specific KIRs in chronic hepatitis patients. Eur. J. Immunol. 42: 447–457. 18. Ray, S. J., S. N. Franki, R. H. Pierce, S. Dimitrova, V. Koteliansky, A. G. Sprague, P. C. Doherty, A. R. de Fougerolles, and D. J. Topham. 2004. The collagen binding a1b1 integrin VLA-1 regulates CD8 T cell-mediated immune protection against heterologous influenza infection. Immunity 20: 167–179.
